Standardization and automization of immunofluorescent analysis across different laboratories using different immunofluorescent equipment and methods represents a significant challenge in the field of bio-assays and diagnostics. The highly sensitive and selective detection and evaluation of fluorescence signals, especially in cell based assays, has lead to the development of fluorescence assays as some of the most important detection methods used in bioanalytics and diagnostics. The advantages of fluorescence detection, for example using fluorescence microscopy, relate to a high sensitivity of detection, relatively simple methods using fluorescence marking of the target objects in cells and the possibility of running parallel analyses of multiple parameters, whereby multiple markings of different target objects with different markers are used. This method therefore replaces more complicated methods such as absorption measurement and complicated and problematic radioactive measurements.
The manual determination and evaluation of indirect immunofluorescence tests (IIF) are however subject to various subjective factors and are additionally influenced by device-specific technical parameters. In addition to the commonly used cell substrates and reagents, microscope technology comprises of fluorescence filters, objectives, light sources, and analysis of the fluorescence patterns to be investigated, whereby each of these factors requires standardization in order to provide reliable and comparable results, especially when considering comparisons between different laboratories with different microscopic equipment and methods.
One example of an immunofluorescence assay that shows strong analytical promise for the health industry, but lacks sufficient standardisation and/or automization between different laboratories is the analysis of DNA damage via detection of DNA double strand breaks (DSBs), of which gamma H2Ax is a known marker.
DNA represents a polymer comprising mononucleotides, which themselves comprise of a base, a deoxyribose sugar and a phosphate group. Nuclear DNA is the universal carrier for genetic information. In the cell nucleus the DNA forms a complex structure with histone proteins, which are present in the form of nucleosomes, which enable the control of various nuclear processes due to the structural arrangement of chromatin. For example, during cell division particular morphological changes occur in chromatin, which allow the formation of condensed chromosomes, which are subsequently essential for reliable chromosome separation to daughter cells. Replication and transcription of DNA are regulated by a complex system of enzymes that must also interact with chromatin in order to carry out their function, which represents the basis for maintaining cellular metabolism. It is essential that the DNA molecule itself and the information coded within remains stable throughout various cellular and nuclear processes, such as transcription, replication and cell division to name but a few.
The DNA of a cell is currently under either direct or indirect influence of various metabolic processes, which can potentially modify the molecular structure of the DNA. Some changes in the structure of DNA are essential for the maintenance or enablement of DNA function, for example the appearance of transient strand breaks during development of antibody producing cells or shortly after replication when individual bases are methylated.
The overwhelming number of such changes, such as for example DSBs, are however not to be seen as important or intended events, but rather as the cause of mutation that is potentially dangerous for the entire organism. Damages to DNA can occur via exogenous processes, for example through ionization, ultraviolet radiation or through mutagenic chemicals, in addition to endogenous processes, for example through metabolic products such as free radicals.
Ionizing radiation can lead to a number of damaging effects in humans, whereby DNA DSBs are one of the most important (1-3). The effect of Ionizing radiation of living tissue is based on the transfer of energy onto the cells, whereby the extent of cell damage depends on multiple factors. Of most importance are the radiation physical properties of the radio nucleides, the absorbed doses, the duration of exposure, the radiation sensitivity of the biological system, the regional energy deposition in addition to the energy density of the radiation (linear energy transfer, LET) (4-7). The survival of a cell depends on the integrity of its DNA, whereby the eradication of malignant cells can occur due to extensive damage to the DNA. As a result of the physical processes a number of chemical changes occur to the DNA, which lead to damage to the DNA and therefore to mutation and potentially death of the cell (8, 9). The most biologically relevant damage in this sense relates to DSBs and single-strand breaks (SSB) of the DNA.
In eukaryotic cells DNA is highly condensed and localized in chromatin structure, whereby the fundamental element of chromatin is the nucleosome, which comprises of 146 DNA base pairs that are wound 1.7 times around the protein core (histone octamer). The protein core of the nucleosome is built of an octamer, which comprises of two of each of the histones H2A, H2B, H3 and H4. Each nucleosome is furthermore bound with an H1 histone, which binds the DNA linking adjacent nucleosomes. Modification of the histones, such as acetylation, deacetylation or phosphorylation, modifies the local chromatin structure and therefore plays a role in the regulation of various nuclear functions, such as replication, transcription or DNA repair.
One example of a histone modification after a DSB is the phosphorylation of histone H2Ax on serine 139. H2Ax that has been phosphorylated on serine 139 is commonly referred to as gamma H2Ax. H2Ax makes up approximately 10% of the H2A population with in chromatin and appears to be evenly distributed. If a DSB occurs then within a few minutes the H2Ax proteins are phosphorylated to form gamma H2Ax. The phosphorylation occurs via protein kinases (ataxia telangiectasia mutated protein (ATM), DNA-dependent protein kinase (DNAPK), ataxia telangiectasia and RAD 3-related protein (ATR) (10-12). The phosphorylation is initiated in the immediate region of the DSB and extends from there, so that eventually H2Ax molecules up to a few megabases from the DSB itself are phosphorylated. The nuclear complexes that can be detected as foci relate to protein complexes that comprise of gamma H2Ax, repair proteins and proteins that function as check point control proteins for the cell cycle.
DNA double-strand breaks can be quantified using a number of methods such as pulse field gel electrophoresis, the comet assay or the tunnel assay. All of these methods exhibit however a relatively low sensitivity, which allows the determination of only a few DSBs per cell. In regards to the comet assay, the threshold at which DSBs can be reliably be distinguished from background lies at radiation doses of approximately 4 Gy. At this radiation dose there are approximately 160 DNA DSBs registered.
Through immunochemical studies it has been shown that quantification of DNA DSBs through measurement of gamma H2Ax foci using immunofluorescence is possible (13). Using this method the number of detectable foci after radiation with heavy ions and after radiation of ionizing photon radiation is proportional to the expected number and expected location of DNA DSBs. This method of quantification of DNA DSBs is sensitive enough so that DNA DSBs can be measured in the micro Gy range. The quantification of foci through staining for serine 139 phosphorylated histone H2Ax (gamma H2Ax) using immunofluorescence represents a relatively new and sensitive method for quantifying a number of DNA DSBs after the effect of ionizing radiation.
The majority of all experiments involving DSB detection using gamma H2Ax involve immunocyto-chemical experiments and evaluation and/or analysis using fluorescence microscopy. Through this approach human mononuclear cells from patients, in addition to cells arising from experimental cell lines, have been tested after being exposed to ionizing radiation. However, the method or determining gamma H2Ax foci using immunofluorescence microscopy is still relatively time consuming, subject to differences in user preferences and technical equipment and is in general a subjective analytical method (14). For the detection of gamma H2Ax foci only manual microscopic approaches have been previously used.
Description of pattern recognition of gamma H2Ax foci using intelligent software algorithms is currently limited to a few academic studies (14, 15). These studies demonstrate the possibility of automatic detection and evaluation of gamma H2Ax foci. However, there exists at the present time no evidence for adapting such algorithms for the automated evaluation of gamma H2Ax foci using commercial setups. Foci (points of light) in the cell must be counted (from 0 to 40) and the results verified through a significant number of tested cells (100 cells per slide). The current developments in immunofluorescent screening are moving towards modular, flexible and compact systems that can be individually configured, which allow the standardization of measurements. Automated interpretation systems, such as AKLIDES, represent fluorescence optical measurement systems that can detect fluorescent signals in a sensitive and meaningful manner. Such systems comprise of motorized inverse fluorescent microscopes, digital cameras, motorized xy-tables, and computers with suitable software for evaluation and analysis. The object to be detected, such as beads or cell structures, can be automatically recognized and described using digital image processing algorithms in a standardized way.
Until now there has been no satisfactory experience or reports as to whether pattern recognition of gamma H2Ax foci using an automatic detection system can be obtained (16). The complications surrounding the automatic pattern recognition of gamma H2Ax foci relates to the complex structures of cell based assays in addition to the complex immunofluorescence patterns generated through such analysis. Furthermore, the calibration, which is of fundamental importance for the standardization of the method, has not been clarified. There exists a serious need for appropriate calibration reagents, which enable the determination and analysis of H2Ax foci using an automated interpretation system.
The technical problem in calibrating gamma H2Ax detection lies in the diversity of immunofluorescence images, which are used for quantification of a signal. Of significant importance is the presence of overlap and layover between separate gamma H2Ax foci. These effects can create significant problems in quantification. Furthermore, the various and differing properties of cell based tests in the systems used to analyse gamma H2Ax foci represent a serious challenge to standardization of the procedure (17, 18).
Automatic microscopic interpretation systems can be standardized using technical components of the analysis system, such as calibrations agents (for example synthetic micropartides), as are commonly used in flow through cytometry.
An intelligent algorithm, which is used for such automatic standardization, is defined by the determining guidelines that it provides (19-21). Automated objective methods are well suited for such standardization, through their exclusion of subjective influencing factors. In this sense, self-learning systems appear upon first inspection to be technically suitable. However, after closer examination it appears that self-learning systems are in fact not suitable for the standardization of the analysis of gamma H2Ax foci. The world wide distribution of devices with self-learning software (especially software that could modify known patterns and also receive input from users regarding new patterns) would improve the individual classification success of each individual laboratory. However, the present situation is that each individual self-learning system tends to develop away from one another, so that the learning processes tend to move further and further away from the original data provided in the system (20, 21). The consequence of this is that self-learning systems provide local (laboratory-specific) improvements without enhancing common inter-laboratory standardization.
A world wide classification standard for gamma H2Ax foci is therefore only possible with a statistically defined method using standardized analytical reference reagents, such as microparticles, to calibrate the method, which then provides a basis for automated optical interpretation.